Pri peptides temporally coordinate transcriptional programs during epidermal differentiation

To achieve a highly differentiated state, cells undergo multiple transcriptional processes whose coordination and timing are not well understood. In Drosophila embryonic epidermal cells, polished-rice (Pri) smORF peptides act as temporal mediators of ecdysone to activate a transcriptional program leading to cell shape remodeling. Here, we show that the ecdysone/Pri axis concomitantly represses the transcription of a large subset of cuticle genes to ensure proper differentiation of the insect exoskeleton. The repression relies on the transcription factor Ken and persists for several days throughout early larval stages, during which a soft cuticle allows larval crawling. The onset of these cuticle genes normally awaits the end of larval stages when the rigid pupal case assembles, and their premature expression triggers abnormal sclerotization of the larval cuticle. These results uncovered a temporal switch to set up distinct structures of cuticles adapted to the animal lifestyle and which might be involved in the evolutionary history of insects.


Fig. S2. Validation of genes upregulated in pri mutant embryos
In situ hybridization confirmed the ectopic expression of 23 genes, selected from their upregulation in transcriptomic data, in pri 1 mutant stage-16 embryos.Scale bar is 100 µm.

Fig. S3. 15 genes were not validated by in situ hybridization
In situ hybridization of genes selected for their up-regulation in pri mutant embryos using transcriptomic data and that do not show obvious changes in their expression pattern between wildtype and pri 1 stage-16 mutant embryos.Scale bar is 100 µm.

Fig. S9. Temporal expression pattern of cuticle genes
Temporal expression of mRNA for cuticle-related genes, as measured in whole wildtype animals from MODENCODE RNA-seq data (2).Relative mRNA levels are indicated as percentage of the highest value for each gene.

GTTGAGACCAAGACTGCCGTGAGC AAGGGCGAGGAG TAGTGGCCTATGCGGCCGCTTACT TGTACAGCTCGTC
Table S5.EST used to generate in situ hybridization for corresponding genes obst-E GH01453

Fig. S4 .
Fig. S4.Role and expression of ken during embryonic development (A) Expression of lcp3, lcp4 and cpr47Eb in stage-16 control embryo and ken 1 and ken 1 /Df(2R)BSC660 mutant embryos, analyzed by in situ hybridization.Scale bar is 100 µm.(B) In situ hybridization in wild type embryos for the ken gene revealing a dynamic expression pattern from early to late embryogenesis.Its expression in the epidermis appears at stage-16.Scale bar is 100 µm.

Fig. S6 .
Fig. S6.pri is required for Ken protein accumulation (A) In situ hybridization for pri and ken mRNA in wild type, ken or pri 1 stage-16 mutant embryos.Scale bar is 100 µm.(B) Images of epidermal cells from embryos expressing GFP-Ken from a Bac construct (ken-GFP.FPTBVK00033) in control and pri 5 mutants.Scale bar is 10 µm.

Fig. S7 .
Fig. S7.Overexpression of ubr3 rescues Lcp4 repression in ecdysone deprived embryos Whole mount in situ hybridization against Lcp4 mRNA in different genetic contexts.Ptc-Gal4 driven overexpression of ubr3 in the epidermis of phm E7 stage-15-16 embryos, which are deprived of ecdysone, is sufficient to repress the ectopic expression of Lcp4.Scale bar is 100 µm.

Fig. S8 .
Fig. S8.Expression pattern of Lcp2, Lcp4 and Cpr47Eb throughout larval stages Lateral view of living larvae expressing Lcp2-prom-eGFP, Lcp4-prom-eGFP and Cpr47ebprom-eGFP at L1, L2 and L3 stages.At late L3 stage, Lcp2 and Lcp4 activities begin and cover the whole epidermis, except in the anal pad region.The activity of Cpr47Eb mainly begins at L3 stage, with intense staining in the anal pad over a faint signal in other epidermal cells.Right panels show higher magnifications of the ventral posterior regions, including the anal pad.In L1 and L2 stages, the contour of larvae (lacking detectable expression) is highlighted by a yellow dotted line.Scale bars are 200 µm.

Fig. S10 .
Fig. S10.Temporal profiles of protein levels for cuticle genesProtein levels defined by quantitative proteomics for cuticle proteins in the entire animal along Drosophila development.Data were extracted from the Butter lab (butterlab.org).

Fig. S11 .
Fig. S11.Expression profile of pri sfGFP during embryogenesis (A) Schematic representation of the pri sfGFP knock-in line.(B) Lateral view of staged embryos (stages 10 to 16) showing the expression of Pri sfGFP during embryogenesis.Nuclei are stained with DAPI (magenta).Scale bar is 100 µm.

Fig
Fig. S12.ken is required for repression of Lcp4 during L1 and L2 larval stages Analysis of Lcp4-prom-eGFP activity in living larvae expressing RNAi ken in epidermal cells, under the control of the epidermal A58-Gal4 driver, at early L1 and L2 larval stages.Scale bar is 200 µm.

Fig. S13 .
Fig. S13.GFP-tagged cuticle proteins accumulate in the apical extracellular space of epidermal cells Localization of GFP-tagged larval cuticle proteins (green) expressed in a subset of epidermal cells, under the control of the Ptc-Gal4 driver.Cpr47Eb, Lcp4 and Lcp2 are distributed in the extracellular space at the apical surface of epidermal cells, similarly to the Zye Zona Pellucida protein (magenta).Black arrows indicate the cell rows expressing Ptc-Gal4.Scale bar is 15 µm.